Abstract

Tolerant self-antigen-specific CD8 T cells fail to proliferate in response to antigen, thereby preventing autoimmune disease. By using an in vivo mouse model, we show that tolerant T cells proliferate and become functional under lymphopenic conditions, even in a tolerogenic environment. However, T cell rescue is only transient, with tolerance reimposed upon lymphorepletion even in the absence of tolerogen (self-antigen), challenging the prevailing paradigm that continuous antigen exposure is critical to maintain tolerance. Genome-wide messenger RNA and microRNA profiling revealed that tolerant T cells have a tolerance-specific gene profile that can be temporarily overridden under lymphopenic conditions but is inevitably reimposed, which suggests epigenetic regulation. These insights into the regulatory mechanisms that maintain or break self-tolerance may lead to new strategies for the treatment of cancer and autoimmunity.

Peripheral tolerant TCRGAG CD8 T cells are phenotypically different and functionally impaired compared with naïve and memory TCRGAG CD8 T cells. (A) Flow cytometric analysis of thymocytes from TCRGAG (naïve) and TCRGAG×Alb:GAG (tolerant) mice. Thymocytes were stained for CD4 and CD8, and single-positive CD8 thymocytes were analyzed for expression of CD44 and TCR by H2-Db/GAG-tetramer binding. (B) Flow cytometric analysis of splenocytes from naïve (gray) and tolerant (black) mice. All CD8+ cells are GAG-tetramer+ and Vα3.2+, and histograms are gated on CD8+ cells. Inset numbers indicate mean fluorescence intensity (MFI). (C) Totals of 106 naïve, 105 memory, or 106 tolerant T cells were transferred intravenously into B6 and/or Alb:GAG hosts and infected 1 day later with 3 × 107 colony-forming units (cfu) of LM-GAG (solid circle) or LM-∅ (open circle) as control. Percent (top) and cell numbers (bottom) of donor T cells in spleens were determined 7 days postinfection. (D) A total of 5 × 105 tolerant T cells (Thy1.2) were transferred intravenously into B6 hosts (Thy1.1), and 3 weeks later mice were infected with 3 × 107 cfu of LM-GAG (solid circle) or control LM-∅ (open circle). At 7 days postinfection, splenocytes were analyzed for cell expansion. All results are representative of at least two or three independent experiments with two to four mice per group. Error bars show SEM. ns, not significant.

Tolerant T cells undergo HP and become functional under lymphopenic conditions, even in a tolerogenic environment. (A) About 2 × 105 to 3 × 105 carboxy-fluorescein-succinimidyl ester (CFSE)–labeled tolerant T cells were transferred (intravenously) into lymphopenic Alb:GAG or B6 WT mice (Thy1.1) 1 day after total body irradiation (TBI; 5Gy). At 11 and 20 days later, CFSE dilution of transferred tolerant T cells was analyzed by flow cytometry. Histograms are gated on CD8+ Thy1.2+ cells. Inset numbers show % of cells that did not proliferate, and numbers of cell divisions are indicated. (B and C) At 22 days after adoptive transfer of 0.5 × 105 to 1 × 105 tolerant T cells into lymphopenic Alb:GAG or B6 hosts, mice were immunized with 3 × 107 cfu of LM-GAG or LM-∅. At 7 days postinfection, peripheral blood was analyzed for expansion of transferred tolerant T cells. Results are representative of at least five independent experiments. ***P < 0.0001. (D) (Top) CFSE-labeled tolerant T cells were transferred into lymphopenic (Thy1.1) Alb:GAG mice, Alb:GAG Ill5−/− mice, or Alb:GAG Ill5−/− mice treated with neutralizing mAb against IL-7 (anti-IL7) 1 day after TBI, and spleen cells were analyzed 12 days later (top). To confirm the efficacy of anti-IL7 treatment, we transferred CFSE-labeled, naïve T cells into lymphopenic B6 wild-type (WI) mice (open histogram) or B6 mice treated with neutralizing anti-IL7 (solid histogram); 12 days later spleen cells were analyzed. Histograms are gated on CD8+ Thy1.2+ cells. (Bottom) At day 22 after transfer, mice were immunized with LM-GAG or LM-∅, and 7 days later spleens were assessed for expansion of donor T cells. Results are pooled from two independent experiments with a total of five to seven mice per group.

Rescued T cells reacquire tolerance in lymphoreplete hosts even in the absence of the tolerogen. (A) Flow cytometric analysis of proliferation of tolerant T cells determined by BrdU incorporation (left) and Ki67 staining (right) 2 to 3 weeks after transfer into lymphopenic mice (top) and at >3 to 4 months after transfer, when recipients had become lymphoreplete (bottom). Histograms are gated on CD8+ Thy1.2+ splenocytes. Percents of BrdU-positive and Ki67-positive transferred T cells are shown. (B) At 3 weeks (HP) or >3 to 4 months (post-HP) after transfer into lymphopenic Alb:GAG or B6 (Thy1.1) mice, mice were immunized with 3 × 107 cfu of LM-GAG or LM-∅. At 7 days postinfection, splenocytes were analyzed for expansion of transferred T cells. Data show mean ± SEM. For B6 hosts, ***P < 0.0001; for GAG hosts, ***P = 0.0007. (C) Intracellular TNF-α and IFN-γ production by tolerant T cells isolated 10 to 14 days (HP) or >3 to 4 months after transfer (post-HP) into irradiated Alb:GAG or B6 hosts. Memory and tolerant T cells are shown as control (top). Results are representative of at least three independent experiments.